There are numerous types of cells that are regularly analyzed for diagnostic and/or research purposes. Human red blood cells are of particular interest. The mature erythrocyte, or red blood cell (RBC), is specialized for the collection and distribution of respiratory gases (i.e., O2 and CO2). The mammalian variety has evolved to the extreme to satisfy this function, having eliminated in the final stages of development in the bone marrow its nucleus and all of its organelles. Instead, the developed RBC acquires a resilient cytoskeleton and is filled with hemoglobin, which comprises one-third of the RBC's mass. Despite the loss of nearly all eukaryotic cellular equipment, it survives a 115 to 120 day circulatory odyssey. At the end of this circulatory period, the senescent RBCs are normally removed from the circulatory system by the spleen and broken down by splenic macrophages. In splenectomized individuals, the task of removing senescent RBCs shifts to the liver.
Analysis of a person's RBCs provides valuable information on the health of that person. However, conventional measurements of red cell status usually provide average values that integrate the entire population of cells into a single value, obscuring critical details. The Reticulocyte count is used to determine the life span of RBCs. Reticulocytes are RBCs that have just entered the circulatory system from the bone marrow. They have not yet discarded all of their messenger RNA, which allows them to be visualized using a nucleic acid stain. If blood cell generation and removal are in steady state equilibrium, the fraction of reticulocytes in the blood will be approximately 0.83%.
Mean corpuscular volume (MCV) measures the average volume of red blood cells, either electronically by a Coulter-type analyzer or calculated from the hematocrit and cell count. Although MCV and cell ages are generally well correlated, some ambiguity remains due to the size heterogeneity of newborn red blood cells. Moreover, this correlation is further influenced by the different rates of volume change, which occur in different phases of the red cell's circulatory lifespan. The size heterogeneity in newborn RBCs derives from the number of cell divisions that occur between a stem cell and a mature erythrocyte. It is necessary to define the population demographics as related to age. It is also important to measure size heterogeneity for other red cell age groups. A concise definition of red cell size heterogeneity will provide important data in the effort to chronicle the progression from red cell birth to senescence in terrestrial and microgravity environments, and will further define the relationship between cell volume and age.
Mean Corpuscular Hemoglobin Concentration (MCHC) measures the concentration of hemoglobin in the cytosol. Red cells continually lose minute amounts of denatured hemoglobin from the moment they enter the circulation. Since red cells have no lysosomes (and only low-capacity proteosomes), the principal hemoglobin losses occur through-extrusion in small membrane vesicles known as Heinz bodies. These vesicles are removed by reticuloendothelial macrophages, most importantly those in the spleen. Water losses compensate for the loss of membrane area. MCHC decreases only modestly throughout the circulatory life of a red cell. This changes more rapidly during the last two to three weeks of red cell circulation, when water losses become accelerated, resulting in red cell dehydration. This dehydration leads to about a 30 percent increase in MCHC, cell density, and an increased cytosolic viscosity for the oldest cells. While red cell cation channels (Gardos channels) are implicated in the accelerated water loss, the importance of these channels and their role in senescent red cell sequestration is not yet fully documented. MCHC and area and volume measurements are needed on single red cells throughout the red cell age spectrum. The whole population average for MCHC and area and volume hides important information about the oldest and the youngest red cell subpopulations.
The micropipetting aspiration procedure of Evans and Waugh yields highly detailed information including volume, surface area, and deformability on individual cells. However, because this-process is extremely labor intensive and slow, it is not practical to apply it to a statistically valid red cell sample, either in a research setting or as a clinical diagnostic tool. Until now, there has not been a satisfactory method to accurately measure the individual cell membrane area for large numbers of red cells.
When taken together, parallel advances in nanofabrication technologies, molecular and cellular biology, biochemistry, and physiology and pathophysiology have created a unique scientific opportunity.
Accordingly, it is an object of the present invention to provide an apparatus and method for rapidly obtaining detailed information including, but not limited to, volume, surface area, and deformability on individual red blood cells.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.